Effects of aerobic training and vitamin D supplementation on glycemic indices and adipose tissue gene expression in type 2 diabetic rats

Type 2 diabetes mellitus (T2DM) is a progressive metabolic disorder mainly caused by overweight and obesity that accumulates pro-inflammatory factors in adipose tissue. Studies have confirmed the efficacy of exercise and vitamin D supplementation in preventing, controlling, and treating diabetes. While, reduced physical activity and vitamin D deficiency are related to increased adiposity, blood glucose level, insulin concentration, and insulin resistance. This study purposed to investigate the effect of 8-week aerobic training with vitamin D supplementation on the expression of AMPK, PGC-1α, and UCP-1 genes expression in the visceral adipose tissue of obese rats with T2DM. In this experimental study, fifty male Wistar rats were divided into 5 groups (n = 10): aerobic training and vitamin D supplementation (AT + Vit D), aerobic training (5 days/week for 8 weeks; AT), vitamin D supplementation (Vit D), diabetic control (C) and NC (Non-Diabetic Control). AT + Vit D and AT groups practiced an 8-week aerobic training, 5 days a week. Vit D and AT + Vit D groups receive 5000 IU of vitamin D by injection once a week while AT and C received sesame oil. After blood sampling, visceral fat was taken to measure AMPK, PGC-1α, and UCP1 gene expression. Data were statistically analyzed by One-way ANOVA and paired sample t-test at a significance level of p < 0.05. Based on our results BW, BMI, WC, visceral fat, insulin, glucose, and HOMA-IR were significantly lower in the AT + Vit D, AT, and Vit D groups compared with the C group (p < 0.01). Furthermore, AT + Vit D, AT, and Vit D upregulated AMPK, PGC-1α, and UCP1 gene expression compared to the C. Based on the results compared to AT and Vit D, AT + Vit D significantly upregulated AMPK (p = 0.004; p = 0.001), PGC-1α (p = 0.010; p = 0.001), and UCP1 (p = 0.032; p = 0.001) gene expression, respectively. Also, AT induced more significant upregulations in the AMPK (p = 0.001), PGC-1α (p = 0.001), and UCP1 gene expression (p = 0.001) than Vit D. Vitamin D supplementation enhanced the beneficial effects of aerobic training on BW, BMI, WC, visceral fat, insulin, glucose, and HOMA-IR in diabetic rats. We also observed that separate AT or Vit D upregulated the gene expression of AMPK, PGC-1α, and UCP1 however, combined AT + Vit D upregulated AMPK, PGC-1α, and UCP1 more significantly. These results suggested that combining aerobic training and vitamin D supplementation exerted incremental effects on the gene expressions related to adipose tissue in animal models of diabetes.


Methods
Ethical approval. All the procedures of the present study were submitted and approved by the Committee of Ethics and Research on the Use of Animals of the Razi University of Kermanshah (IR.RAZI.REC.1401.013) and experiments were performed following the specific Iranian laws on the Bioethics in Experiments with Animals (nº 11.794/2008) and complied with the ARRIVE guidelines for the Care and Use of Laboratory Animals.
Animals. In this experimental study, fifty 4-5-week-old male Wistar rats weighing 180 ± 10 g were selected and divided into two main groups; diabetic (n = 40 rats) and Non-Diabetic Control (NC; n = 10 rats) groups. After inducing diabetes through intraperitoneal streptozotocin (STZ) and nicotinamide with a High-Fat Diet (HFD), diabetic rats were randomly assigned to 4 groups (10 in each group); aerobic training + vitamin D supplementation (AT + Vit D), aerobic training (AT), vitamin D supplementation (Vit D), control (C). AT and AT + Vit D groups practiced an 8-week aerobic training, 5 days a week. Vit D and AT + Vit D groups receive 5000 IU of vitamin D by injection once a week. While, AT and C were injected with sesame oil instead. This placebo-controlled randomized and single-blinded study was registered in the Iranian registry of clinical trials at http:// www. irct. ir: number, IRCT20220510054809N1. Also, the Research Ethics Committee of Razi University approved and supervised the study design and conduction (no. IR.RAZI.REC.1401.013), Iran. Animal care, maintenance and sacrifice have been conducted according to the Danish "Animal Welfare Act" (LBK 1343 of 04/12/2007). www.nature.com/scientificreports/ Obesity induction method. Two weeks prior to the commencement of the interventional program rats were acclimatized to the new environment. Then, male rats were fed high-fat diet to increase weight in the form of pellets (purchased from Beh-Parvar Company); a mixture of standard mouse food powder (365 mg/kg), sheep fat (310 mg/kg), mixed vitamins and minerals (60 mg/kg), dl-methionine (3 mg/kg), yeast powder (1 mg/kg), and chloride sodium (1 mg/kg). Animals were kept in transparent polycarbonate cages with a length of 30 cm, and width and height of 15 cm. Animals were maintained under a 12-h light/dark cycle, temperature of 25 ± 2 °C, and 45-55% humidity. All animals had free access to special food and water in a 500 ml bottles 16,24 ; the ethical principles of working with laboratory animals were considered in the present study.
Diabetes induction method. After increasing weight (more than 300 g) following 2 weeks, 60 mg/kg BW streptozotocin (dissolved in 0.1 M citrate buffer with pH = 4.5) and then, after 15 min, 110 mg/kg BW of nicotinamide were intraperitoneally injected to induce diabetes in male Wister rats. To ensure the induction of T2DM, blood sugar was measured 2 weeks after the injection with the help of a glucometer and blood samples of the lateral tail vein after immersing in 42 °C water for 40-50 s; Blood sugar above 200 mg/l was considered as an indicator of diabetes 25 . Animals received no insulin treatment during the study.
Intervention programs. Aerobic training protocol. After the induction of diabetes, the rats were placed in 4 groups of 10 randomly and also through weight equalization. One week before the start of the main aerobic training sessions rats ran on the treadmill for 5 min at a speed of 8-10 m/min, 5 days a week to get familiar with the treadmill. The aerobic training program consisted of 8 weeks of running on a zero-incline treadmill, at 15-25 m/min, for 30-60 min, 5 days a week. In the first week, rats performed 30 min of aerobic training on a treadmill at a speed of 15 m/min. Then, the exercise intensity and duration increased gradually to reach 25 m/ min and 60 min in the 8th week. The training intensity was equal to 50-60% of VO 2max 26 . Animals also warmed up and cooled down for 10 min at 5 m/min before and after the aerobic training. To stimulate stopped rats to run, a combination of sound (hitting the wall of the rotating tape) and low voltage electric stimulations were used. In the first week, the low voltage electric stimulus was used along with the sound stimulus. Then, only the sound stimulus was used considering the ethical guidelines of working with laboratory animals 16,24 (Fig. 1).

Vitamin D supplementation.
In the present study, Vit D and AT + Vit D groups received 5000 international units (IU) of vitamin D per week by single-dose injection. AT and C were injected with sesame oil instead of vitamin D. Vitamin D serum concentration was measured using a vitamin D enzyme-linked immunosorbent assay (ELISA) rat kit (immune diagnostics system Ltd, Boldon, UK; with intraassay coefficient of variation = 1.63%, and sensitivity of method = 1.33 mg/dL) 22 . www.nature.com/scientificreports/ Food intake, bodyweight, and body mass index. All animals were weighed weekly, between 09:00 and 11:30 a.m. using a scale. Body length (nose-to-anus) and Body Mass Index (BMI) were calculated. Food Intake (FI) was measured by subtracting the weight of the uneaten food from the total equal amount of food (20 g/d) given in each cage.
Tissue and blood sampling. In order to eliminate the acute effects of training and uncontrollable variables caused by the training program, 48 h after the last training session, the animals were anesthetized (46,15) by intraperitoneal injection of a combination of ketamine (70 mg/kg BW) and xylazine (3-5 mg/kg BW) considering the ethical principles. The blood sample was taken from the vena cava; The plasma concentrations of glucose and insulin were measured by the Hitachi Auto Analyzer (type 7170; Hitachi Electronics, Hitachi, Japan). The Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) was calculated using the concentration of glucose and insulin as follows: Visceral adipose tissue was immediately separated and weighed. After washing with saline, to prevent RNA degradation, it was placed in tubes containing RNA Later, transferred to liquid nitrogen, and then stored in a refrigerator at − 80 °C for further measurements.
RNA extraction/real-time PCR. RNA isolation from 20 mg of adipose tissue was performed using the combined protocol of TRI Reagent ® (MRC Inc., US) and miRNeasy methods (Viragene, Iran). First, the frozen tissue was homogenized as recommended by the manufacturer using a gentleMACS™ Octo Dissociator system, M tubes (Viragene, Iran), and the RNA_02 program in 2 ml of TRI Reagent ® buffer. After incubating at room temperature for 5 min, the samples were centrifuged at 12,000g at 4 °C for 10 min. The desired layer was piped into a 1.5 ml tube, mixed with 400 μl of chloroform, and centrifuged at 12,000g at 4 °C for 30 min after keeping the sample for 3 min at room temperature. Then the upper layer was mixed with ethanol by inverting the tube several times and loaded on the miRNeasy spin column (Viragene, Iran) performing the manufacturer's protocol. Finally, RNA was eluted in 30 μl of RNAse-free water. Reverse transcription into cDNA was performed using 1 μg of total RNA with a Prime Script RT reagent kit (Viragene, Iran). Quantitative RT-PCR was performed using TB Green Premix Ex-Taq II (TaKaRa, Dalian, China). Beta-2-Microglobulin (B2M) was used as the reference gene to measure relative gene expression. The results were evaluated by using 2 −ΔΔCt comparative method and Light Cycler SW1.1 software. The sequence of the primers is shown in Table 1. The Viragene kit, Iran was used to measure AMPK gene expression with a sensitivity of 0.021; to measure PGC-1α gene expression Viragene mini kit50, Iran was used with a sensitivity of 0.06; And to measure UCP-1 gene expression, Viragene kit, Iran was used with a sensitivity of 0.06.

Results
Mean body weight, BMI, and food intake (FI) are shown in Table 2. There was a significant difference between groups in mean BW, BMI, and FI after 8-week of intervention; BW, BMI, and FI reduced significantly in the AT + Vit D, AT, and Vit D groups at the end of the study compared with the beginning (p < 0.01). The highest reduction in BW, BMI, and FI was in the AT + Vit D group, whereas mean BW, BMI, and FI increased significantly in the C and NC groups.  Table 3 shows, there was a statistically significant difference between groups in visceral fat, insulin, glucose, and HOMA-IR; with the lowest level in AT + Vit D and the highest in the C group. While, the results show a statistically significant difference between groups in serum 25-hydroxyvitamin D; with the highest level in AT + Vit D and the lowest in the C group. Based on the results, the mean visceral fat, insulin, glucose, and HOMA-IR were significantly lower in the AT + Vit D, AT, and Vit D than in the C group. In addition, significant differences were observed in visceral fat, insulin, glucose, HOMA-IR, and serum 25-hydroxyvitamin D between the NC group and other groups (p < 0.05 for all three variables).
The results of AMPK, PGC-1α, and UCP1 gene expression are shown in Figs. 2, 3, 4. There was a significant difference in AMPK, PGC-1α, and UCP1 gene expression between the diabetic and NC groups at the end of the intervention. One-way ANOVA showed a significant difference in Akt, PEPCK, and G6Pase gene expression between diabetic groups. Furthermore, AT + Vit D, AT, and Vit D upregulated AMPK, PGC-1α, and UCP1 compared to the C. Based on the results, AT + Vit D significantly upregulated AMPK (p = 0.004; p = 0.001), PGC-1α (p = 0.010; p = 0.001), and UCP1 (p = 0.032; p = 0.001) compared to AT and Vit D, respectively. Also, AT induced more significant upregulations in the AMPK (p = 0.001), PGC-1α (p = 0.001), and UCP1 gene expression (p = 0.001) than Vit D.

Discussion
In the present study, we initially demonstrated that separate aerobic training and vitamin D improved body weight and serum parameters such as fasting blood glucose, insulin, and HOMA-IR. However, better results were observed when combining aerobic training and vitamin D. Our results were consistent with those of previous studies indicating the same improvements following aerobic training 27,28 . Also, studies showed that 8 weeks of Vitamin D supplementation reduced body weight, BMI, visceral fat, waist circumference, serum Table 2. Comparison of mean ± SD of body weight, BMI, FI, and WC before and after intervention. AT + Vit D Aerobic Training + Vitamin D Supplement, AT Aerobic Training, Vit D Vitamin D Supplement, C Control, NC Non-Diabetic Control. Data analysis was done by the analysis of one-way analysis of variance test followed by post hoc Tukey's test; p † : Statistical analysis was done by paired sample t-test. *Significantly different in comparison pre and post within the groups. ¥ Significantly different comparing Δ between groups. µ Significantly different compared to AT. € Significantly different compared to Vit D. α Significantly different compared to C. β Significantly different compared to Sham.

Variables
AT + Vit D (n = 10) AT (n = 10) Vit D (n = 10) C (n = 10) NC (n = 10) p-value a  Table 3. Comparison of mean ± SD of visceral fat, insulin, glucose, HOMA-IR, and vitamin D after the intervention among the groups. AT + Vit D Aerobic Training + Vitamin D Supplement, AT Aerobic Training, Vit D Vitamin D Supplement, C Control, NC Non-Diabetic Control. One-way ANOVA test followed by Tukey's posthoc test. Dissimilar letters represent a significant difference between the groups, the level of significance was set at 5% (p < 0.05). ¥ Significantly different comparing Δ between groups.  29,30 . Vitamin D is a hormone that is synthesized in the skin and plays a vital role in maintaining optimal body function, particularly in the regulation of calcium homeostasis and bone health. The level of vitamin D in the body is influenced by various factors, including dietary intake, sun exposure, and physical activity 31 . The cellular and molecular mechanism of the observed differences in vitamin D values can be traced to the effect of exercise-induced changes on vitamin D metabolism 32 . During physical activity, there is an increase in the circulating levels of parathyroid hormone (PTH), a hormone that plays a crucial role in the regulation of calcium and phosphate homeostasis 33  Moreover, exercise stimulates the increase in vitamin D receptor (VDR) expression in muscle cells as well as the synthesis of vitamin D binding protein (DBP) in the liver, which facilitates the transport and uptake of vitamin D into the muscles 34 . These changes ultimately result in an increase in the intracellular concentration of 1,25(OH)2D in the muscle fibers, which contributes to the beneficial effects of exercise on muscle health and function 27 .

Variables AT + Vit D (n = 10) AT (n = 10) Vit D (n = 10) C (n = 10) NC (n = 10) p-value
However, the observed differences in vitamin D values may have been influenced by exercise-induced changes in vitamin D metabolism, as well as variations in initial levels. Understanding the cellular and molecular mechanisms of these factors can help researchers design more effective interventions to optimize vitamin D status and promote overall health. Therefore, it is crucial to control for these factors in any study examining the relationship between exercise and vitamin D status.
The molecular mechanisms of aerobic training in the improvement of diabetes might include the upregulation of insulin transporters in the cell membrane, reduction of adipokines, inflammatory and oxidative stress  41 . It seems that increased intracellular regulatory factors (such as PGC-1α) following aerobic training and vitamin D supplementation might have altered the UPC-1 expression in this study. Furthermore, studies showed that increased secretion of hormones (e.g. thyroid hormones 42 , norepinephrine 43 , and Irisin 44 ) during aerobic training might upregulate the UCP-1 gene expression in adipose tissue through different mechanisms. Moreover, a wide spectrum of studies support the increment of PGC-1α genes following aerobic training in skeletal muscle of humans 45 and visceral adipose tissue of obese rats 16 . However, the observed weight loss in this study might be due to the reduced food intake following aerobic training and vitamin D supplementation. According to body weight and food intake results obtained in this study, the most significant reduction was in the group with aerobic training combined with vitamin D. The mechanism by which an increase in the concentration of vitamin D in the body weight and food intake affects body composition is difficult to explain. However, some studies reported no effect from vitamin D on body weight change and energy expenditure and little improvement in cardiovascular risks with cholecalciferol 46,47 . In agreement with our study, Hoseini et al. reported that vitamin D supplementation combined with aerobic training significantly improves BW and FI in ovariectomized rats 22 . Also, the decreased appetite hormone, and increased AMPK gene expression, and PGC-1α/UCP-1 pathway following aerobic training might be among the possible mechanisms. In addition, a proposed model related to the relationship between vitamin D deficiency and more food intake is that the decrease in the concentration of calcidiol flow in the hypothalamus induces an increase in the set point of body weight. On the other hand, the appetite increases and the energy consumption decreases through activating the hypothalamic NPY/AgRP neurons and POMC/CART inhibiting. Furthermore, the results of the present study showed that AMPK, PGC-1α, and UCP1 expression increased after 8 weeks of vitamin D supplementation. Recent evidence has demonstrated that vitamin D is a potential stimulating factor in increasing AMPK activity and gene expression 48,49 . Gauthier et al. observed low AMPK activation in inflamed adipose tissue of obese human participants 50 . Chang investigated the role of vitamin D in oxidative stress and mitochondrial changes suggesting that the observed potent inhibitory effect of 1,25(OH)2D on muscle oxidative stress and mitochondrial dynamics might be at least involved in the activation of AMPK 49 . A recent research also studied the effects of vitamin D supplementation on adipose tissue inflammation and NF-κB/AMPK activation in obese mice fed a high-fat diet indicating a significant increase in AMPK activity by   51 . Additionally, vitamin D is believed to regulate lipolysis dependent on its localization; stimulating lipogenesis in subcutaneous tissue but inhibiting it in visceral adipose tissue 52 . Vitamin D supplementation was demonstrated to increase the expression of genes such as PGC-1α and PDK4 and thereby enhance the oxidation of fatty acids 53 . Vitamin D has long been known as a hormonal regulator of the cAMP signaling pathway 54 . In turn, cAMP activates cAMP-dependent PKA, the endocrine system, and AMPK activity 55 which phosphorylates and activates lipolysis enzymes and leads to cascades of phosphorylation that stimulate the activity of PGC1α, PPARα, and UCP1 56,57 . The results also showed a significant increase in the gene expression of AMPK, PGC-1α, and UCP1 in WAT in AT + Vit D compared to the other three groups (Vit D, AT, and control). However, the main mechanism for combined AT + Vit D is not elucidated, it might be a better approach in inducing AMPK, PGC-1α, and UCP1 expression in WAT of T2DM rats.
Strength and limitations of the study. Our study had several strengths using a randomized, placebocontrolled trial with no dropouts and evaluating the gene expression alterations in animal model of diabetes. However, it is premature to conclude a definitive answer. Also, not evaluating the levels of vitamin D before and after the induction of diabetes or at the beginning of the study and other important transcriptional and posttranscriptional factors were among the limitations of the present study.

Conclusion
Vitamin D supplementation enhanced the beneficial effects of aerobic training on BW, BMI, WC, visceral fat, insulin, glucose, and HOMA-IR in diabetic rats. We also observed that separate AT or Vit D upregulated the gene expression of AMPK, PGC-1α, and UCP1 however, combined AT + Vit D upregulated AMPK, PGC-1α, and UCP1 more significantly. These results suggested that combining aerobic training and vitamin D supplementation exerted incremental effects on the gene expressions related to adipose tissue in animal models of diabetes.

Data availability
The datasets generated and analysed during the current study are not publicly available due to ongoing data analysis but are available from the corresponding author on reasonable request.